Population structure of community-acquired extended-spectrum beta-lactamase producing Escherichia coli and methicillin resistant Staphylococcus aureus in a French region showed no difference between urban and rural areas

Antimicrobial resistance is a global health issue and extended-spectrum β-lactamase producing Escherichia coli (ESBL-Ec) and methicillin-resistant Staphylococcus aureus (MRSA) are of particular concern. Whole genome sequencing analysis of isolates from the community is essential to understand the circulation of those multidrug-resistant bacteria. Our main objective was to determine the population structure of clinical ESBL-Ec and MRSA isolated in the community setting of a French region. For this purpose, isolates were collected from 23 sites belonging to 6 private medical biology laboratories in the Bourgogne-Franche-Comté region. One hundred ninety ESBL-Ec and 67 MRSA were sequenced using the Illumina technology. Genomic analyses were performed to determine the bacterial typing, presence of antibiotic resistance genes, metal resistance genes as well as virulence genes. Analysis showed that ST131 was the major ESBL-Ec clone circulating in the region, representing 42.1% of the ESBL-Ec isolates. The blaCTX-M genes represented 98% of blaESBL with the majority being blaCTX-M-15 (53.9%). MRSA population consisted of mainly of CC8 (50.7%) and CC5 (38.8%) clonal complexes. Interestingly, we found a prevalence of 40% of the zinc resistance gene czrC in our MRSA population. We observed no differences in our ESBL-Ec or MRSA populations between urban and rural areas in our French region, suggesting no impact of population density or rural environment.


Introduction
Multidrug-resistant bacteria (MDRB) are a major public health problem, responsible for increased hospitalization, morbidity and mortality [1].Two MDRB, Escherichia coli and Staphylococcus aureus, account for much of the burden of multi-drug resistant bacterial infections, which caused 30,000 deaths in Europe in 2015 [2].Both these MDRB were initially described in the hospital setting but have therefore spread in the community.Methicillin-resistant Staphylococcus aureus (MRSA) was first described in 1961 [3].By the early 1980s, MRSA had spread worldwide and in some countries was responsible for the majority of S. aureus infections in hospitals [4].MRSA infections initially affected the elderly, children and immunocompromised hospitalized patients.Moreover, in the 1990s, community-acquired MRSA (CA-MRSA) clones emerged and spread widely in the United States, infecting healthy individuals with no history of hospitalization [5].In France, the main hospital-acquired MRSA (HA-MRSA) clones were ST8 and ST5, while ST80 was mainly community-acquired [6,7].
Gram-negative multidrug-resistant bacteria and in particular extended-spectrum β-lactamase (ESBL)-producing Enterobacterales are a growing public health problem in Europe [8].The first ESBLs described in 1983 were derived from TEM and SHV enzymes and were produced by Enterobacterales responsible for hospital outbreaks.ESBLs carrying bla CTX-M genes were described in France in 1991 in hospitals, but then spread massively into the community.Escherichia coli producing ESBL (ESBL-Ec) ST131 is now the most important community clone in Europe and worldwide [9].It has been observed that most of the hospital-acquired ST131 infections are acquired in the community [10].
In France, a laboratory network monitors antimicrobial resistance in the community [11], but the genomic data available to characterize circulating clones in France are rather rare and outdated [6].The community setting is composed of rural and urban areas.The One Health approach suggests that the spread and mode of acquisition of MDRB may differ between urban and rural settings [12,13].However, the available studies on this particular topic were conducted in Africa or South-East Asia and do not reflect European lifestyles [14][15][16].
Our main objective was to perform an epidemiological and genomic study of ESBL-Ec and MRSA isolates responsible for community-acquired infections in Bourgogne Franche-Comte ŕegion (France).The secondary objective was to compare these multi-resistant strains between rural, semi-urban and urban contexts.

Study design
The study was approved by the local ethic committee before the study commencement considering that no patients data were collected, and samples were anonymised.We conducted a prospective multicenter study including 23 sampling sites belonging to 6 medical biology laboratories in the Bourgogne-Franche-Comte ´region (France).Laboratories sent to a central laboratory (in the University Hospital of Besanc ¸on) all isolates identified as ESBL-Ec or MRSA collected in the first three months of 2019 and 2021 from clinical samples of community patients (only one isolate per patient).Isolates were then categorized according to their origin (urban area >100,000 inhabitants, semi-urban area between 20,000 and 100,000 inhabitants and rural area if < 20,000 inhabitants).The authors did not have access to any information that could identify individual participants during or after data collection.

Microbiological analysis
Presumptive ESBL-Ec and MRSA were cultured on chromogenic agar plate (URI4 urine, bio-Me ´rieux, Marcy-L'Etoile, France) and Muller-Hinton agar (Mast Diagnostic, Amiens, France), respectively.Identification was confirmed by MALDI-TOF MS (Microflex LT, Bruker Daltonik GmbH, Bremen, Germany) according to the manufacturer's recommendations (log � 2 and repeatability � 3).Production of ESBL for E. coli and resistance to methicillin for MRSA were checked according to EUCAST 2022 guidelines.Determination of antibiotic resistance to other clinically used antibiotics was also performed according to EUCAST 2022 guidelines.

Statistics
Fisher's exact test or Chi2 test were used for categorical variables and the Wilcoxon-Mann-Whitney test or Kruskal-Wallis test for continuous variables as appropriate.The p values for multiple comparisons were adjusted using the Holm-Bonferroni method.For all analyses, a p value <0.05 was considered statistically significant.Analyses were performed using R studio.

Escherichia coli-Population structure and antibiotic susceptibility
Among the 190 ESBL-Ec included, phylogroup B2 was predominant with 62.2% of the isolates, phylogroups D and A were represented by 12.1% and 10.0% of the isolates, respectively.We identified 42 different STs among which ST131 predominated (42.1%), the other four most frequently represented STs were ST10, ST69, ST88, and ST1193 with 6.8%, 5.3%, 5.3%, and 5.3% of the isolates, respectively.We observed no difference between 2019 and 2021 for phylogroups

Staphylococcus aureus-Population structure
Among the 67 MRSA strains included, we identified nine different STs clustered into six clonal complexes (CCs) (S1 Fig) .CC8 and CC5 were largely predominant with 34 (50.7%) and 26 (38.8%) isolates, respectively.spa typing showed a predominance of spa t008 associated to CC8 and t777 associated to CC5 with, respectively, 17 (25.4%)and 18 (26.9%)strains (Fig 3).All of our MRSA strains carried the mecA gene.The SCCmec type IV cassette was the most frequent (68.6%) followed by the type VI cassette (29.9%) and by the type V cassette (1.5%).CC8 carried SCCmec type IV (n = 33) or SCCmec type V (n = 1) cassettes.CC5 carried SCCmec type VI or type IV for 6 and 20 strains, respectively.We observed no difference between 2019 and 2021 for the complex clonal (p = 0.107) or the SCCmec cassette (p = 0.096) in our MRSA population.

Staphylococcus aureus-Resistance to antibiotics and virulence
blaZ gene was present in 54 strains (80.1%) without linkage to CC.The proportion of acquired resistance genes and/or mutations conferring resistance is shown in Fig 4 .Two strains (ST8 t121 and ST88) expressed the lukF and lukS genes responsible for the production of Panton-Valentine leukocidin.

Staphylococcus aureus-Metal resistance
All isolates carried copA gene as well as ars operon that encode copper and arsenic resistance, respectively.Twenty-eight strains (41.8%) possessed the zinc resistance gene czrC (encoding a heavy metal translocating P-type ATPase), for which we identified two alleles with a nucleotide homology of 78%.Twenty-one strains carried the czrC_1 gene (KF593809.1)and seven strains carried the czrC_2 gene (CP030605.1).One CC8 t008 strain carried the mer operon for mercury resistance and the cad operon for cadmium resistance.The czrC_1 gene were present in the SCCmec type VI cassette of all CC5 t777 strains (n = 18).The czrC_1 gene was also found in the SCCmec type IV cassette of two CC8 t008 strains and in the SCCmec type VI cassette of one CC5 t2379 strain.The presence of the czrC_1 gene in the SCCmec type IV cassette was associated with the additional presence of the ccrA4 and ccrB4 genes between the orfX region and the IS431 insertion sequence.The sequences ccrB4-ccrA4-czrC_1 had a nucleotide homology of more than 99% between CC5 t777 and CC8 t008.The 7 czrC_2 genes were present in the SCCmec type IV cassette in six CC8 t008 strains and in one CC398 strain.

Discussion
In this community-based study on the scale of a French region, whole genome sequencing of a significant set of ESBL-Ec and MRSA revealed that ESBL-Ec population is largely dominated by the ST131 clone and bla CTX-M genes and that MRSA belongs to 2 major clones whose origin is probably the hospitals.National surveillance data for the region [31] suggests that our collection is representative since it represents approximately 60% of ESBL-Ec and MRSA isolated during the study period.These results are consistent with most European surveillance studies that include genotyping [32][33][34].We also found similar population structures of ESBL-Ec and MRSA between, urban, semi-urban or rural areas suggesting no impact of population density or rural environment on the epidemiology of these MDRB.
Although bla ESBL genes are mostly borne on plasmids (especially for bla CTX-M-1 ), chromosomal integration is frequent for bla CTX-M-14 and bla CTX-M-15 genes.The localization of 12.6% of our bla ESBL gene could not be identified, probably due to the size of the contigs.Chromosomal integration of bla CTX-M has already been observed [35] and could be beneficial for the propagation of the Ec-ESBL as in Klebsiella pneumoniae [36].
Co-resistance associated with ESBL genes is a major problem for patient treatment.More than 40% of the strains present in our study are co-resistant to fluoroquinolones and cotrimoxazole.These resistances are particularly problematic for the management of pyelonephritis and male urinary tract infections, which require the prescription of carbapenems or intravenous injection of antibiotics several times a day [37].Several studies have revealed that the bla OXA-1 gene is responsible for increased MICs of piperacillin/tazobactam (TZP) and may be responsible for increased mortality [38,39].We identified a prevalence of 33% in our community ESBL-Ec strains, associate to a resistance to TZP of 20% in our Ec-ESBL population.This resistance rate is similar to that of French hospital strains [40].Probabilistic monotherapy with TZP in patients at high risk of ESBL could expose to a considerable risk of failure.
We identified in our ESBL-Ec strains a significant increase in co-resistance to cotrimoxazole, tetracyclines and macrolides in urban areas.These co-resistant strains mainly harbored mph (A), dfrA17 and tet(A) as already observed in E. coli ST131 [41].Tetracycline consumption has been identified as a significant cause of ESBL acquisition [42] One hypothesis is that the use of tetracyclines selected the strains resistant to tetracyclines, cotrimoxazole and macrolides.
MRSA isolated in our community share the characteristics of MRSA isolated in French hospital.Indeed, CC5 t777 (New pediatric clone) and CC8 t008 (Lyon Clone) represent together more than 50% of the MRSA isolated in our study.Strains considered as CA-MRSA are rare, with only one strain USA300 (ST8 t121, SCCmec IV, PVL+) and one ST88 strain that produced PVL [43].The USA300 clone is endemic in the United States but remains rare in France.No ST80 PVL-producing strain (European clone) was found in our study although it has been identified as the major CA-MRSA strain in Europe [44].ST398 has emerged in the last two decades to take an important place in the livestock-associated MRSA population.Nevertheless, only 2 MRSA ST398 strains were identified in our study, in contrast with the high proportion of MSSA ST398 recovered in our region [45].
We identified the presence of zinc resistance genes in more than 40% of our human MRSA population.Zinc resistance was initially associated to LA-MRSA, notably ST398.The high prevalence of czrC in our study is surprising given that MRSA ST398 strains [46] represent only 3% of our population.These czrC-containing strains can be separated into two distinct populations: CC5 SCCmec VI czrC_1 and CC8 SCCmec IV czrC_2 strains.The presence of czrC_1 genes in two CC8 t008 SCCmec IV strains is likely explained by the integration of a ccrA4-ccrB4-czrC_1 sequence similar to our CC5 t777 SCCmec VI strains.This suggests a high mobilization capacity of the czrC_1 gene.Recently, a 22% prevalence of czrC was observed in ST5 SCCmec II strains of human origin in the United States [47].Our study is the first, to our knowledge, to identify a dissemination of two distinct lineages of MRSA carrying two different czrC alleles.
Our study has several limitations.First, we did not recover the origin of the sample, but it is likely that the majority of ESBL-Ec were from urine samples while MRSA were from skin samples.Second, the comparison between urban, peri-urban, and rural areas implies that people have little contact between areas and that they go to their local laboratories for testing, which we were unable to verify.Thirdly, the patients were all living at home at the time of sampling, but we did not have information on hospitalization history, which can explain the carriage of MRSA of hospital origin [48].
In conclusion, our results provide additional data for monitoring the evolution of MDRB in the community sector, in which genomic data are quite rare.Our data confirm previous data.We did not highlight differences between urban and rural strains.
(p = 0.36), sequence type (p = 0.153) and for the ESBL gene (p = 0.092) in our Escherichia coli strains.The population structure of ESBL-Ec is shown in Fig 1 and the phenotypic resistance results displayed in Fig 2. Eighty ESBL-Ec strains (42.1%) were co-resistant to cefotaxime, ciprofloxacin, and cotrimoxazole.ST131 strains were more resistant to gentamicin (p = 0.01) and to ciprofloxacin (p <0.001) than the strains of other STs.

Fig 1 .
Fig 1. Phylogenetic tree of 190 clinical ESBL-producing Escherichia coli strains isolated in a French region generated by approximatelymaximum-likelihood and rooted on Escherichia fergusonii.The major STs are represented by a specific color on the dendrogram, as defined in the legend.The innermost colored ring indicates the phylogroup, while the outermost colored ring indicates the geographical origin of the sample.https://doi.org/10.1371/journal.pone.0294433.g001

Fig 2 .
Fig 2. Comparison of 190 clinical ESBL-producing Escherichia coli strains isolated in a French region according to their geographical origin.A. Distribution of strains according to their phylogroup.B. Phenotypic resistance of the strains.C. Distribution of the number of virulence genes per strain.D. Distribution of the number of antibiotic classes with at least one detected gene per strain.E. Distribution of the number of quinolone resistance determining regions (QRDR) per strain.F. Proportion of strains with acquisition of at least one antibiotic resistance gene per strain.Significant differences are indicated by asterisks (*: p < 0.05; **: p < 0.01).CTX, cefotaxime; FEP, cefepime; CAZ, ceftazidime; AMC, amoxicillin/ clavulanic acid; CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; GEN, gentamicin; TZP, piperacillin/tazobactam; FOX, cefoxitin; AMK, amikacin.https://doi.org/10.1371/journal.pone.0294433.g002